Nuclear Medicine and Biology 40 (2013) 625–629

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Nuclear Medicine and Biology
journal homepage: www.elsevier.com/locate/nucmedbio

Predominant contribution of L-type amino acid transporter to
4-borono-2- 18F-ﬂuoro-phenylalanine uptake in human glioblastoma cells☆
Mitsuyoshi Yoshimoto a,⁎, Hiroaki Kurihara b, Natsuki Honda b, Keiichi Kawai c, Kazuyo Ohe c,
Hirofumi Fujii a, Jun Itami d, Yasuaki Arai baDivision of Functional Imaging, National Cancer Center Hospital East, Chiba
Diagnostic Radiology, National Cancer Center Hospital, Tokyoc
Faculty of Health Sciences, Kanazawa University, IshikawadRadiation Oncology, National Cancer Center Hospital, Tokyoba r t i c l e

i n f oArticle history:
Received 24 January 2013
Received in revised form 15 February 2013
Accepted 18 February 2013
Keywords:
18
F-FBPA
BNCT
Glioblastoma
Amino acid transporters
System La b s t r a c t
Introduction: 4-Borono-2- 18 F-ﬂuoro-phenylalanine (18 F-FBPA) has been used to anticipate the therapeutic
effects of boron neutron capture therapy (BNCT) with 4-borono-L-phenylalanine (BPA). Similarly, L[methyl- 11C]-methionine (11C-MET), the most popular amino acid PET tracer, is a possible candidate for thispurpose. We investigated the transport mechanism of 18 F-FBPA and compared it with that of 14C-MET inhuman glioblastoma cell lines.
Methods: Uptake of 18 F-FBPA and 14C-MET was examined in A172, T98G, and U-87MG cells using 2aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (a system L-speciﬁc substrate), 2-(methylamino)-isobutyricacid (a system A-speciﬁc substrate), and BPA. Gene expression was analyzed by quantitative real timepolymerase chain reaction.
Results: System L was mainly involved in the uptake of 18 F-FBPA (74.5%–81.1% of total uptake) and 14C-MET(48.3%–59.4%). System A and ASC also contributed to the uptake of 14C-MET. Inhibition experiments revealed
that BPA signiﬁcantly decreased the uptake of 18 F-FBPA, whereas 31%–42% of total 14C-MET uptake wastransported by BPA non-sensitive transporters. In addition, 18 F-FBPA uptake correlated with LAT1 and total
LAT expressions.
Conclusion: This study demonstrated that 18 F-FBPA was predominantly transported by system L in humanglioblastoma cells compared to 14C-MET. Although further studies are needed to elucidate the correlation
between 18 F-FBPA uptake and BPA content in tumor tissues, 18 F-FBPA is suitable for the selection of patients
who beneﬁt from BNCT with BPA.
© 2013 Elsevier Inc. All rights reserved.

1. Introduction
Boron neutron capture therapy (BNCT) is radiotherapy using highlinear energy transfer (LET) alpha particle ( 4He) and recoiling lithium( 7Li), which are produced by a nuclear ﬁssion reaction of 10B with low
energy thermal neutrons. Clinical interest in BNCT has focused
primarily on patients with high grade glioma [1–3]. The success of
BNCT requires sufﬁcient accumulation of 10B in cancer tissues. The
representative 10B carrier used in clinical trials is 4-borono-Lphenylalanine (BPA), and a preferable tumor to normal tissue ratio
is greater than 3–5 [4]. Therefore, estimation of 10B content in cancer
tissue helps to anticipate the therapeutic potential of BNCT.
An 18 F-labelled analog of BPA, 4-borono-2- 18 F-ﬂuoro-phenylalanine ( 18 F-FBPA) has been developed to predict 10B concentration in☆ The authors have no conﬂicts of interest to declare.
⁎ Corresponding author. National Cancer Center Hospital East, 6-5-1, Kashiwanoha,
Kashiwa, Chiba 277-8577, Japan. Tel.: +81 4 7134 6832; fax: +81 4 7134 6832.
E-mail address: miyoshim@ncc.go.jp (M. Yoshimoto).
0969-8051/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.nucmedbio.2013.02.010

tumors [5,6]. Imahori et al. indicated that the 10B concentration andkinetics of BPA can be estimated using the rate constants of 18 F-FBPApositron emission tomography (PET) [7,8]. 18 F-FBPA and BPA are
thought to be transported via system L, which recognizes neutralamino acids with large, branched, or aromatic side chains likephenylalanine as substrates. Thus, four isoforms of system L, LAT1,
LAT2, LAT3, and LAT4, have been identiﬁed so far [9–12]. In particular,
overexpression of LAT1 is widely found in many tumors [13,14]. Dettaet al. found that the uptake of BPA in human brain tumor samples wasinhibited by phenylalanine and 2-aminobicyclo-(2.2.1)-heptane-2carboxylic acid (BCH), indicating the signiﬁcant involvement ofsystem L [15]. However, the contribution of other transporters, suchas system A and ASC, in the uptake of BPA was not investigated. Thus,
the transporter systems involved in the uptake of 18 F-FBPA in glioma
cells still remain to be determined.
18
F-FBPA is synthesized only in certain hospitals, thus, its utility
seems clinically challenging. However, L-[methyl- 11C]-methionine( 11C-MET), which is the most popular amino acid PET tracer, available
in many hospitals, is also a candidate for screening of patients

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M. Yoshimoto et al. / Nuclear Medicine and Biology 40 (2013) 625–629

applicable to BNCT with BPA. PET with 11C-MET is useful for diagnosisof brain tumors [16,17]. Moreover, 11C-MET is also transported into
cells by system L [18,19]. To determine the difference between the
two PET tracers, we investigated the transport mechanism of 18 FFBPA and 14C-MET using three human glioblastoma cell lines. Directcomparison of both tracers gives us valuable information to estimate
the therapeutic beneﬁts of BNCT with BPA.

control (100%). System A and ASC transport was calculated bysubtracting the tracer uptake in the presence of MeAIB fromuptake in the Na +-PBS control and Na +-free PBS control,
respectively. System L transport was calculated by subtracting
tracer uptake in the presence of BCH from the uptake in the Na +free PBS control. Finally, System PAT transport was calculated bysubtracting the tracer uptake in the presence of MeAIB from the
Na +-free PBS control.

2. Materials and methods
2.1. Cell culture

2.4. qRT-PCR analysis

Three human glioblastoma cell lines used were A172, T98G, (DSPharma Biomedical Co., Ltd., Osaka, Japan) and U-87 MG (American
Type Culture Collection, Manassas, VA, USA). A172 cells were cultured
in Dulbecco’s modiﬁed Eagle’s medium (4.5 g/L glucose; Invitrogen,
Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS).
T98G and U-87MG were cultured in Eagle’s minimum essential
medium (Invitrogen) supplemented with 10% FBS. All cell lines weremaintained in a humidiﬁed atmosphere of 5% CO2 in air at 37 °C.

Total RNA was extracted from A172, T98G, and U-87MG cells
using an RNeasy Mini kit (QIAGEN, Tokyo, Japan). cDNA wassynthesized from total RNA using an AfﬁnityScript QPCR cDNA
Synthesis kit (Agilent Technologies, Santa Clara, CA, USA). The
thermal proﬁle of the reverse transcription was as follows: 5 min
at 25 °C, 15 min at 42 °C for and 5 min at 95 °C. System A(SNAT1, SNAT2, SNAT4) and ASC (ASCT1, ASCT2) as Na +dependent transporters, system L (LAT1, LAT2, LAT3, LAT4) asNa +-independent transporter, and 4F2hc which forms heterodimers with LAT1 and LAT2 were analyzed. Glyceraldehyde-3phosphate dehydrogenase (GAPDH) and β-actin (ACTB) wereused as internal controls. Primers for each target gene weresynthesized by Nihon Gene Research Laboratories (Table 1; Miyagi,
Japan). Quantitative real time polymerase chain reaction (qRTPCR) was performed on an Mx3005P (Agilent Technologies) using
Brilliant III Fast SYBR Green QPCR Master Mix (Agilent Technologies). The thermal proﬁle of QPCR reaction was as follows: 3 min
at 95 °C for 1 cycle, 5 s at 95 °C followed by 20 s at 60 °C for
40 cycles, 1 min at 95 °C followed by 30 s at 55 °C and 30 s at
95 °C for 1 cycle. The mRNA copy number was calculated fromstandard curves generated by amplifying serial dilutions of aknown concentration of puriﬁed amplicons. Expression data werenormalized against the average copy number of housekeeping
genes. All samples were analyzed in triplicate.

2.2. Synthesis of 18 F-FBPA
18

F-FBPA was synthesized by direct electrophilic radioﬂuorinationof BPA (Sigma-Aldrich, St. Louis, MO, USA) using 18 F-acetyl hypoﬂuorite as described previously [20]. Puriﬁcation of 18 F-FBPA was
performed by HPLC using YMC-Pack ODS-A column (20 × 150 mm;
YMC, Kyoto, Japan) eluted with 0.1% acetic acid at a ﬂow rate of 10 ml/
min. The radiochemical purity was determined by HPLC. The
radiochemical purity and the speciﬁc activity of 18 F-FBPA were
N 99.5% and 25 MBq/μmol, respectively.
2.3. In vitro uptake of 18 F-FBPA and 14C-MET in human glioblastoma
cell lines
In vitro uptake studies were carried out as described previously[21]. L-[methyl- 14C] methionine ( 14C-MET) was purchased from
American Radiolabeled Chemicals Co. (St. Louis, MO, USA). Tumor
cells (1 × 10 5 cells/well) were seeded in 24-well plates. The uptake
studies were performed on the subsequent day after seeding. The
sodium-containing assay buffer was composed of phosphate-buffer
saline (Na +-PBS) supplemented with 137 mM NaCl, 2.7 mM KCl,
8 mM Na2HPO4, 1.5 mM KH2PO4, 5.6 mM D-glucose, 0.9 mM CaCl2,
and 0.5 mM MgCl2. In the sodium-free assay buffer (Na +-free PBS),
NaCl and Na2HPO4 were replaced by choline chloride and K2HPO4,
respectively. Preliminary experiments to determine the time curse of
18
F-FBPA uptake into T98G cells indicated that the uptake waslinearly dependent on incubation time up to 30 min (data notshown). Thus, the uptake of 18 F-FBPA and 14C-MET was measured
for 30 min. After removing culture medium, the cells were preincubated with 500 μl of the assay buffer for 10 min at 37 °C. The cells
were then incubated with 500 μl of the assay buffer containing 18 FFBPA (185 kBq) or 14C-MET (18.5 kBq) for 30 min at 37 °C. Afterincubation, the cells were washed twice with ice-cold assay buffer anddissolved in 0.1 N NaOH. The radioactivity in the cells was measured
with a gamma counter (AccuFLEX γ7001; Aloka, Tokyo, Japan) and aliquid scintillation counter (Tri-Carb 3110TR; PerkinElmer, Waltham,
MA, USA).
To characterize the transport system, we performed theinhibition experiment with inhibitors: 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (BCH; Sigma-Aldrich) for system L,
2-(methylamino)-isobutyric acid (MeAIB; Sigma-Aldrich) for system A, and BPA at concentrations indicated in ﬁgures. To calculate
the contribution of amino acid transporter systems, we used themethods reported by Kobayashi et al. [18]. In brief, uptake of 18 FFBPA and 14C-MET in the absence of inhibitors was used as the

Table 1
Sequences of primers.
Ofﬁcial Symbol

Alias

PrimerSequence

SLC38A1

SNAT1

SLC38A2SNAT2SLC38A4SNAT4SLC1A4ASCT1

SLC1A5ASCT2SLC7A7y+LAT1

SLC7A6y+LAT2SLC7A5LAT1

SLC7A8LAT2SLC43A1

LAT3

SLC43A2LAT4

SLC3A2

4F2hc

ACTB

actin, beta

GAPDH

-

Forward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
Reverseattttgggactcgcctttg
agcaatgtcactgaagtcaaaagt
cctatgaaatctgtacaaaagattgg
ttgtgtacccaatccaaaacaa
tgttctggtcatccttgtgc
aaaactgctggaagaataaaaatcag
tttgcgacagcatttgctac
gcacttcatcatagagggaagg
gaggaatatcaccggaacca
aggatgttcatcccctcca
cctgcttatatccaggaccaa
ggccacttcatactcagtgct
cctatccctgctttactgttcaa
aagctgaagtagttgataagctgga
gtggaaaaacaagcccaagt
gcatgagcttctgacacagg
ttgccaatgtcgcttatgtc
ggagcttctctccaaaagtcac
tttggtggcatctgcctaa
attaacgtggagcgcaggt
cagggagaccctctgtgg
cggtagcagatcaggtagagc
taccggggtgagaactcgt
cagccaaaactccagagcat
ccaaccgcgagaagatga
ccagaggcgtacagggatag
agccacatcgctcagacac
gcccaatacgaccaaatcc

M. Yoshimoto et al. / Nuclear Medicine and Biology 40 (2013) 625–629

627

Fig. 1. Contribution of amino acid transporters to 18 F-FBPA (left) and 14C-MET (right) uptake in human glioblastoma cell lines. 18 F-FBPA or 14C-MET was incubated with cells for
30 min (n = 4).

2.5. Statistical analysis
The correlations between tracer uptake and gene expression wereanalyzed using GraphPad Prism v5.04 (Graph Pad Software Inc., San
Diego, CA, USA).
3. Results
3.1. Competitive inhibition study of 18 F-FBPA and 14C-MET
We estimated the contribution of each amino acid transportsystem to 18 F-FBPA and 14C-MET in A172, T98G, and U-87MG cells(Fig. 1). System L was predominantly involved in tumor uptake of
18
F-FBPA (74.5%–81.1% of total uptake). Contribution of system ASC
and other transport systems in the uptake of 18 F-FBPA was 5.6%–
21.6% and 1.9%–6.9%. Uptake of 14C-MET was also mainly mediated bysystem L (48.3%–59.4%). In addition, 28.8% of total 14C-MET uptake in
U-87MG cells was mediated by system ASC. In T98G cells, system A
contributed to 22.7% of total 14C-MET uptake. Contribution of PAT,
system ASC, and system A was 8.1%–12.1% in A172 cells.
3.2. Dose-dependent inhibition of FBPA and MET uptake by BPA
Uptake of 18 F-FBPA was dose-dependently inhibited by BPA in all
cell lines (Fig. 2). BPA (1 mM) reduced the uptake of 18 F-FBPA to
2.1%–7.1% of control. On the other hand, the uptake of 14C-MET was
decreased by 1 mM BPA to 31.2% in A172, 42.2% in T98G and 32.4% in
U-87MG.

3.3. Gene expression of amino acid transporters in A172, T98G
and U-87MG
The gene expression of amino acid transporters was summarizedin Table 2. The Na +-dependent transporter systems, system A, andsystem ASC, were predominantly expressed in T98G cells. The
expression of system ASC in U-87MG cells was higher than that in
A172 cells. Among system L, LAT1 was highly expressed in T98G cells,
while LAT4 was expressed in A172 cells.
3.4. Relationship between tracer uptakes and gene expressions
Fig. 3 showed that the uptake of 18 F-FBPA correlated with LAT1
and total LAT expression (r = 0.8576 for LAT1 and 0.9418 for total
LAT). The uptake of 14C-MET correlated with total LAT expression(r = 0.6691), but not with LAT1 expression. However, these
correlations are statistically not signiﬁcant.
4. Discussion
We investigated and compared the transport mechanism of 18 FFBPA and 14C-MET in human glioblastoma cell lines, A172, T98G, andU-87MG. The present study revealed that although system L is a main
contributor for the uptake of 18 F-FBPA and 14C-MET, other transporters also mediate the uptake of 14C-MET.
BCH drastically inhibited the uptake of 18 F-FBPA in all glioblastoma cell lines, suggesting the major involvement of system L,
containing LAT1, LAT2, LAT3, and LAT4. The uptake of BPA and 125I-2iodo-L-phenylalanine was inhibited by BCH and phenylalanine,Fig. 2. Effect of BPA to 18 F-FBPA (left) and 14C-MET (right) uptake in human glioblastoma cell lines. 18 F-FBPA or 14C-MET was incubated with the indicated concentration of BPA andthe cells for 30 min (n = 4).

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M. Yoshimoto et al. / Nuclear Medicine and Biology 40 (2013) 625–629

Table 2
Expression of amino acid transporters in human glioblastoma cell lines.

Na+-dependent

SystemFamilyA172

T98G

U-87MG

A

SNAT1
SNAT2
SNAT4
ASCT1
ASCT2
yLAT1
yLAT2
LAT1
LAT2
LAT3
LAT4
4F2hc

14.14
39.31
0.18
4.60
10.10
ND
8.75
15.67
1.67
1.66
42.59
37.21

21.50
105.84
ND
10.70
88.86
0.88
10.89
59.01
0.64
1.66
9.19
191.79

24.23
41.79
ND
12.09
41.84
ND
11.94
16.19
2.44
0.49
3.17
101.10ASC
y +L
Na+-independent

L

Coupling factor

4F2hc

Data are expressed as copy number per 1000 copies of housekeeping genes.
ND: not determined.

supporting our results [15,22,23]. However, these studies havefocused only on LAT1 because its expression is elevated in a varietyof tumors, and LAT4 has not yet been well-characterized in tumor[14]. The qRT-PCR analysis revealed that 18 F-FBPA uptake demonstrated an enhanced correlation with total LAT, mainly LAT1 and LAT4,
than LAT1 alone. Haase et al. reported higher expression of LAT4 than
LAT1 in HT-29 colon cancer cells and FaDu head and neck cancer cells[24]. Phenylalanine is a substrate for LAT4 as well as LAT1 [10]. These
data imply that LAT4 expression could be an important factor for 18 FFBPA uptake in tumors.
Similarly, system L was predominantly involved in the uptake of
14
C-MET. Unlike 18 F-FBPA; however, other components, such assystem ASC and system A, also contributed to the uptake of 14C-MET.
This resulted in a weak correlation between 14C-MET uptake and total
LAT expression, even though one of the reasons may be because of thesmall number of cell lines used in this study. Soriano-Garcia et al.
indicated the involvement of four systems containing system L in METtransport [25]. Shotwell et al. reported that the uptake of MET in
Chinese hamster ovary (CHO) cells was mediated by system L (51%),
system ASC (22%), and system A (9%) [26]. Thus, uptake of 14C-MET isregulated by a diverse expression of amino acid transporters.
Other reason for this weak correlation might be because theincorporation of 14C-MET into protein contributes to the uptake of
14
C-MET. 14C-MET is metabolized and then incorporated to protein.
The reﬂux and the metabolism of 14C-MET through the incubation for
30 min would not be negligible. Therefore, the uptake of 14C-MET
might not solely reﬂect the transport activity of 14C-MET.
Our inhibition study and other previous studies have indicatedthat 14C-MET was partly transported by BPA-non-sensitive transporters. Therefore, 11C-MET-PET may overestimate the concentrationof BPA in tumor tissues because of its compatibility with a broad rangeof amino acid transporters. In this study, SNAT2, ASCT2, and system L
were expressed at various levels in glioblastoma cell lines. The
expression of these transporters was remarkably elevated in humancancers [14,21]. Moreover, MET is a better substrate for ASCT2 andSNAT2 than phenylalanine [27,28]. These data suggest that 11C-MET
would be preferentially taken up in tumors with high expression of
ASCT2 and/or SNAT2.
System L-speciﬁc imaging agents may be possible candidates toselect patients with the therapeutic beneﬁts of BNCT with BPA. Our
results indicated that 18 F-FBPA is a system L-speciﬁc imaging agent.
Uptake of phenylalanine derivatives such as 3-O-methyl-6- 18 Fﬂuoro-L-dopa and 123/125I-2-iodo-L-phenylalanine was mediated bysystem L [22–24]. Wiriyasermkul et al. reported that L-3- 18 F-αmethyl tyrosine is selectively transported by LAT1 but not by LAT2,
whereas the contribution of LAT4 is unclear [29].
Based on our results, 18 F-FBPA is a selective substrate for system L
containing LAT1 and LAT4 in three human glioblastoma cell lines.
Other amino acid transporters such as system ASC and system A partly
contributed to the 14C-MET uptake compared with 18 F-FBPA. Further
studies are needed to investigate the correlation between the tracer
uptake and BPA contents in tumor tissues for selecting patients who
may beneﬁt from BNCT with BPA.

Acknowledgments
This study was supported in part by the National Cancer Center
Research and Development Fund.

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Fig. 3. Correlation between tumor uptake of 18 F-FBPA (A) and 14C-MET (B) and theexpression of amino acid transport system genes. 18 F-FBPA positively correlated with
LAT1 and total LAT (r = 0.8576, p = 0.3440 for LAT1 and r = 0.9418, p = 0.2183 fortotal LAT). Although there was no correlation between 14C-MET uptake and LAT1
(r = −0.1497, p = 0.9043), total LAT had a weak correlation with 14C-MET (r =0.6691, p = 0.5334).

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